Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays

Cell Mol Life Sci. 2019 Mar;76(6):1185-1199. doi: 10.1007/s00018-018-03003-2. Epub 2019 Jan 8.


Receptor tyrosine kinases (RTKs) play key roles in various aspects of cell biology, including cell-to-cell communication, proliferation and differentiation, survival, and tissue homeostasis, and have been implicated in various diseases including cancer and neurodevelopmental disorders. Ligand-activated RTKs recruit adapter proteins through a phosphotyrosine (p-Tyr) motif that is present on the RTK and a p-Tyr-binding domain, like the Src homology 2 (SH2) domain found in adapter proteins. Notably, numerous combinations of RTK/adapter combinations exist, making it challenging to compare receptor activities in standardised assays. In cell-based assays, a regulated adapter recruitment can be investigated using genetically encoded protein-protein interaction detection methods, such as the split TEV biosensor assay. Here, we applied the split TEV technique to robustly monitor the dynamic recruitment of both naturally occurring full-length adapters and artificial adapters, which are formed of clustered SH2 domains. The applicability of this approach was tested for RTKs from various subfamilies including the epidermal growth factor (ERBB) family, the insulin receptor (INSR) family, and the hepatocyte growth factor receptor (HGFR) family. Best signal-to-noise ratios of ligand-activated RTK receptor activation was obtained when clustered SH2 domains derived from GRB2 were used as adapters. The sensitivity and robustness of the RTK recruitment assays were validated in dose-dependent inhibition assays using the ERBB family-selective antagonists lapatinib and WZ4002. The RTK split TEV recruitment assays also qualify for high-throughput screening approaches, suggesting that the artificial adapter may be used as universal adapter in cell-based profiling assays within pharmacological intervention studies.

Keywords: Cell-based assay; Lapatinib; Receptor tyrosine kinases; Split TEV recruitment assay; TEV protease.

MeSH terms

  • A549 Cells
  • Acrylamides / metabolism
  • Acrylamides / pharmacology
  • Animals
  • Biological Assay / methods*
  • Cell Line, Tumor
  • ErbB Receptors / antagonists & inhibitors
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism
  • GRB2 Adaptor Protein / genetics
  • GRB2 Adaptor Protein / metabolism*
  • Humans
  • Lapatinib / metabolism
  • Lapatinib / pharmacology
  • PC12 Cells
  • Protein Binding / drug effects
  • Pyrimidines / metabolism
  • Pyrimidines / pharmacology
  • Rats
  • Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Reproducibility of Results
  • src Homology Domains*


  • Acrylamides
  • GRB2 Adaptor Protein
  • Pyrimidines
  • Lapatinib
  • WZ4002
  • ErbB Receptors
  • Receptor Protein-Tyrosine Kinases